Type 2 diabetes (T2D) is associated with chronic inflammation that predominantly originates from immune system cells. These cells are chronically activated by the in vivo milieu of T2D patients, which includes elevated plasma levels of free fatty acids (FFAs) and endotoxin. Both of FFAs and endotoxin are ligands for toll-like receptors (TLRs), and both generally activate pro-inflammatory cytokine production upon TLR engagement. These cytokines likely play important roles in the chronic systemic inflammation characterizing T2D and other metabolic syndromes. Multiple studies show that controlling inflammation in obese mice by blocking TLR function decreases the incidence of insulin resistance, a hallmark of T2D. Although many cell types are sources of inflammatory cytokines, immune cell cytokines are required for insulin resistance. Taken together, these analyses indicate that TLR-activated inflammation from immune system cells is a key process that promotes T2D and other metabolic diseases. Although monocytes are generally considered the most important immune cell type for pro-inflammatory cytokine production, B cells also produce significant levels of cytokines. Our new data on B cells from T2D patients demonstrate that B cell TLR2 and TLR4 activation generally results in production of pro-inflammatory cytokines. However, TLR4 ligand decreases TLR2- mediated production of TNF-1. This anti-inflammatory effect may be balanced by the complete inability of B cells from T2D patients to secrete IL-10, a critical anti-inflammatory cytokine. The discoveries of an elevated percentage of TLR4-positive B cells in T2D patients and the mixed inflammatory responses of these B cells to TLR ligands lead to the hypothesis that B cells and B cell TLRs play important roles in T2D by regulating inflammation thus insulin resistance and glucose tolerance. Because the general endotoxemia and elevated free fatty acids in T2D patients provide innumerable TLR ligands, our preliminary data predict that B cell TLRs influence T2D through cytokine regulation in vivo. Although additional studies on human B cell TLR function are likely to be mechanistically and clinically important, the next critical step in this line of investigation is to unequivocally demonstrate B cells and B cell TLRs play roles in T2D inflammation and pathogenesis. These studies will be undertaken in a diet-induced obesity mouse model of T2D that takes advantage of available genetically altered mice. This project will test the effects of 1. the absence of B cells;2. TLR function only on B cells;and 3. B cell-specific TLR inactivation on inflammatory and metabolic outcomes of high fat diet in vivo. These studies will define the role of B cells in metabolic imbalance, and predict the likely efficacy of using approved B cell ablation therapies as new treatments for metabolic syndrome and T2D. PUBLIC HEALTH RELEVANCE: Type 2 diabetes (T2D) is increasing at epidemic rates world-wide, and the treatment arsenal for this disease is limited. Inflammation plays an important role in the etiology and complications of T2D. Inflammation stemming from immune cell cytokine production plays a demonstrated role in insulin resistance, one of the key features of T2D. The role of myeloid cell cytokines in T2D is relatively well understood. Our new work in T2D patients demonstrates that B cells may be another major source of cytokines in T2D. However, our human subjects research, by its nature, cannot definitively link B cells to disease pathogenesis. The project proposes to test the role of B cells and their surface receptors in T2D using model organisms. Importantly, this work will determine whether existing B cell ablation therapies can be used as fundamentally new treatments for T2D.